Method for treating amyloid disease

ABSTRACT

Disclosed herein are methods for treating amyloid disease in humans by clearing amyloid peptides from one or more bodily fluids such as, e.g. blood, of a patient. In particular, the methods are based on the administration of compounds capable of binding to amyloid-beta (Aβ) or on dialysis of blood or plasma exchange in order to remove Aβ peptides from the blood circulation, and/or brain or other affected organs.

This application is a divisional of U.S. patent application Ser. No.10/540,294, filed Jun. 20, 2005, which is the U.S. National Stage ofInternational patent application number PCT/US2003/040744, filed Dec.18, 2003, which claims the benefit of U.S. provisional application No.60/434,736, filed Dec. 19, 2002, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods for treating human amyloiddisease. Specifically, this invention relates to methods of reducing thelevels of amyloid-beta (Aβ) peptides in bodily fluids by, e.g., theadministration of compounds capable of associating with Aβ, or thedialyzation of blood through a column or membrane to remove free Aβ.

BACKGROUND OF THE INVENTION

Amyloid disease (disorders of protein folding), or amyloidosis, ischaracterized by the accumulation of a peptide, including the Aβpeptide, existing as abnormal insoluble cross-β sheet fibrils or amyloiddeposits in the affected organs. Amyloid diseases include, but are notlimited to, Alzheimer's disease, type 2 diabetes, Huntington's disease,Parkinson's disease, and chronic inflammation. Amyloidosis is also acommon and serious complication of long-term heamodialysis for end-stagerenal failure. Amyloidosis—in which amyloid deposits are the directcause of death—is responsible for about one per thousand of all deathsin developed countries.

Alzheimer's Disease (AD) is the most common form of late-life dementiain adults (Ghiso et al., Adv. Drug Deliv. Rev. 2002; 54(12):1539-51),constituting the fourth leading cause of death in the United States.Approximately 10% of the population over 65 years old is affected bythis progressive degenerative disorder that is characterized by memoryloss, confusion and a variety of cognitive disabilities.

Neuropathologically, AD is characterized by four major lesions: a)intraneuronal, cytoplasmic deposits of neurofibrillary tangles (NFT), b)parenchymal amyloid deposits called neuritic plaques, c) cerebrovascularamyloidosis, and d) synaptic and neuronal loss. One of the key events inAD is the deposition of amyloid as insoluble fibrous masses(amyloidogenesis) resulting in extracellular neuritic plaques anddeposits around the walls of cerebral blood vessels. The majorconstituent of the neuritic plaques and congophilic angiopathy is Aβ,although these deposits also contain other proteins such asglycosaminoglycans and apolipoproteins.

Evidence that amyloid may play an important role in the earlypathogenesis of AD comes primarily from studies of individuals affectedby the familial form of AD (FAD) or by Down's syndrome. Down's syndromepatients have three copies of the APP gene and develop AD neuropathologyat an early age (Wisniewski et al., Ann Neurol 1985; 17:278-282).Genetic analysis of families with hereditary AD revealed mutations inchromosome 21, near or within the Aβ sequence (Ghiso et al., Adv. DrugDeliv. Rev. 2002; 54(12):1539-51), in addition to mutations within thepresenilin 1 and 2 genes. Moreover, it was reported that transgenic miceexpressing high levels of human mutant APP progressively developamyloidosis in their brains (Games et al., Nature 1995; 373:523-527).These findings appear to implicate amyloidogenesis in thepathophysiology of AD. In addition, Aβ fibrils are toxic to neurons inculture, and to some extent when injected into animal brains (Sigurdssonet al., Neurobiol Aging 1996; 17:893-901; Sigurdsson et al., JNeuropathol Exp Neurol 1997; 56:714-725).

Furthermore, several other pieces or evidence suggest that thedeposition of Aβ is a central triggering event in the pathogenesis ofAD, which leads subsequently to NFT formation and neuronal loss. Theamyloid deposits in AD share a number of properties with all the othercerebral amyloidoses, such as the prion related amyloidoses, as well asthe systemic amyloidoses. These characteristics are: 1) being relativelyinsoluble; 2) having a high degree of 5-sheet secondary structure, whichis associated with a tendency to aggregate or polymerize; 3)ultrastructurally, the deposits are mainly fibrillary; 4) the presenceof certain amyloid-associating proteins such as amyloid P component,proteoglycans and apolipoproteins; and 5) deposits show a characteristicapple-green birefringence when viewed under polarized light after Congored staining.

The same peptide that forms amyloid deposits in the AD brain was alsofound in a soluble form (sAβ) normally circulating in human body fluids(Seubert et al., Nature 1992; 359:325-327; Shoji et al., Science 1992;258:126-129). sAβ was reported to pass freely from the brain to theblood (Ji et al., Journal of Alzheimer's Disease 2001; 3:23-30; Shibataet al., J Clin Invest 2000; 106:1489-99; Ghersi-Egea et al., J Neurochem1996; 67(2):880-3; Zlokovic et al., Biochem Biophys Res Commun 1994;205:1431-1437), reported that the blood-brain barrier (BBB) has thecapability to control cerebrovascular sequestration and transport ofcirculating sAβ, and that the transport of sAβ across the BBB wassignificantly increased in guinea pigs when sAβ was perfused as acomplex with apolipoprotein J (apoJ). The sAβ-apoJ complex was found innormal cerebrospinal fluid (CSF, Ghiso et al., Biochem J 1993;293:27-30; Ghiso et al., Mol Neurobiol. 1994; 8:49-64) and in vivostudies indicated that sAβ is transported with apoJ as a component ofthe high density lipoproteins (HDL) in normal human plasma (Koudinov etal., Biochem Biophys Res Commun 1994; 205:1164-1171). It was alsoreported by (Zlokovic et al., Proc Natl Acad Sci USA 1996;93:4229-4234), that the transport of sAβ from the circulation into thebrain was almost abolished when the apoJ receptor, gp330, was blocked.It has been suggested that the amyloid formation is anucleation-dependent phenomena in which the initial insoluble “seed”allows the selective deposition of amyloid (Jarrett et al., Cell 1993;73:1055-1058; Jarrett et al., Biochemistry 1993; 32:4693-4697).

Therapeutic strategies proposed for treating Alzheimer's disease andother amyloid diseases include the use of compounds that affectprocessing of the amyloid-β precursor protein (Dovey et al., JNeurochem. 2001; 76:173-182), or that interfere with fibril formation orpromote fibril disassembly (Soto et al., Nat Med 1998; 4:822-826;Sigurdsson et al., J Neuropath Exp Neurol 2000; 59:11-17; and Findeis MA., Biochim Biophys Acta 2000; 1502:76-84), as well as theadministration of Aβ antibodies to disassemble flibrillar Aβ, maintainAβ solubility and to block the toxic effects of Aβ (Frenkel et al., JNeuroimmunol 1999; 95:136-142). However, recently a Phase II clinicaltrial using a vaccination approach where Aβ1-42 was injected intoindividuals in the early stages of Alzheimer's disease was terminatedbecause of cerebral inflammation observed in some patients.

Thus, despite these advances in the art, to date, there is no cure oreffective therapy for reducing a patient's amyloid burden or preventingamyloid deposition in AD. Moreover, even the unequivocal diagnosis of ADcan only be made after postmortem examination of brain tissues for thehallmark neurofibrillary tangles (NFT) and neuritic plaques. Thus, thereexists a need in the art for developing effective methods for reducing apatient's amyloid burden.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that amyloiddiseases can be treated by removing Aβ peptides from a patient's bodilyfluids. This can be accomplished by the administration of compounds thatassociate with Aβ in order to bind to free Aβ. Free Aβ can also beremoved from a patient's bloodstream by dialysis. Both methods lead toan efflux of Aβ from the affected organs, resulting in the reduction ofa patient's amyloid burden.

In one aspect the present invention provides a method for treating apatient suffering from an amyloid disease comprising administering to apatient in need of such treatment a therapeutically effective amount ofa compound that is capable of associating with Aβ. In one embodiment,the compound binds to free Aβ present in the blood. In anotherembodiment, the compound binds to free Aβ present in the blood and inthe brain. For example, the compound can be administered intravenously,and the transport of the compound into the brain enhanced by treatingthe patient in a manner so as to permeabilize the blood-brain-barrier.In a preferred embodiment, the blood-brain barrier is selectivelypermeabilized to compounds capable of associating with Aβ, includinglarge protein ligands to Aβ and their complexes with Aβ.

In another aspect, the present invention provides a method for treatinga patient suffering from an amyloid disease comprising filtering theblood of said patient through a filter, membrane or column with which isassociated a compound capable of binding Aβ, thereby removing Aβ fromthe patient's blood.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the following description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show binding curves for the interactions of apoE3 and apoE4with immobilized Aβ40 and Aβ42 peptides, FIGS. 1A-1C and 1D-1F,respectively. (A, D) Binding of Sf9-derived apoE3 and apoE4 indelipidated form or incorporated into r-HDLs. (B, E) Binding ofHEK-derived apoE3 and apoE4 in delipidated form or in native HDLparticles. (C, F) Binding of RAW-derived apoE3 and apoE4 in delipidatedform or in native HDL-particles. Each point represents the mean(±standard deviation) of triplicates. See Example 1.

FIG. 2 shows that transgenic mice immunized with K₆Aβ1-30(E₁₈E₁₉) hadsignificantly fewer errors in the radial arm maze compared to theirvehicle-treated controls (two-way ANOVA, repeated measures, p<0.05).

FIG. 3 depicts that (A) K₆Aβ1-30(E₁₈E₁₉) immunization induced asubstantially more pronounced IgM response compared to IgG response asdetected in plasma at 1:500 dilution; and that (B) the IgM antibodiesgenerated cross-reacted with Aβ1-40.

FIGS. 4A-4F show that (A, B, C) K₆Aβ1-30(E₁₈E₁₉) immunizationpreferentially reduced small (34% reduction, p=0.02) and medium sizedplaques (29% reduction, p=0.04), and that (D, E, F) low amyloid plaqueburden correlated with high IgM levels against the immunogen (p<0.05)and Aβ1-42 (p=0.05). A trend for correlation was seen for IgMrecognizing Aβ1-40.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for removing the Aβ moleculefrom blood, from a blood component such as plasma or serum, and/or fromanother bodily fluid than blood.

In one embodiment, the Aβ peptide is removed from blood ex vivo with nouse of any Aβ-ligand. For example, patient's blood can be subjected toconvective dialysis, using an unidirectional membrane with a cut-offweight higher than that of Aβ, or to plasma exchange, thereby replacingplasma containing Aβ with Aβ-free plasma.

In another embodiment, the Aβ, present in the blood or blood component,is contacted ex vivo with an agent capable of associating with orbinding to Aβ, i.e., an Aβ ligand. Such ligands include anti-Aβantibodies or Aβ-binding antibody fragments, as well as theAβ-associating agents described in Table 1. The ligand can be attachedto a solid support or present in a dialysis compartment not allowingbound Aβ to flow back into the plasma compartment. For example, anAβ-ligand attached to a solid support can be incorporated in ahemofiltration device or other device known in the art. In thisembodiment, Aβ is “specifically removed”, meaning that all or a portionof the amount of target molecules removed from the blood or bloodcomponent without the removal of other peptides, hormones or other bloodconstituents, and the patient is not exposed directly to the ligand byinjection or other means.

Methods of ex vivo treatment include, but are not limited to, plasmaperfusion, hemodialysis, and hemofiltration. The methods can beconducted on a continuous or batch basis. “Cleansed” blood or bloodcomponent may be returned to the patient concurrently with perfusiontreatment or following perfusion treatment. The perfused blood or bloodcomponent may be supplemented or reconstituted with components fromdonated blood, artificial or synthetic components, therapeutic agents,or some combination thereof.

In yet another embodiment, the contacting between Aβ and an Aβ-ligandsuch as an anti-Aβ antibody or Aβ-binding fragment thereof, or one ofthe Aβ-associating agents described in Table 1, may take place in vivoafter administering the Aβ-ligand to a patient. Typically, the ligand isadministered in a manner so that it reaches the blood circulation,binding to circulating Aβ. The Aβ-ligand pair can thereafter be clearedfrom the blood and excreted or degraded via, e.g., hepatic or renalcatabolism. In a particular embodiment, the Aβ-ligand is also capable ofcrossing the blood-brain barrier to some or a significant degree intothe cerebroventricular fluid (CVF) in the brain. After binding tosoluble Aβ in the CVF, the Aβ-ligand pair can diffuse or be transportedinto blood, from which it is cleared in the same manner described above.For example, compounds such as insulin-like growth factor I (IGF-I), orother compounds having a similar effect, can be used to selectivelypermeabilize the blood-brain-barrier to transport of Aβ-ligands orcomplexes between Aβ and a ligand. IGF-I have been shown to induceclearance of Aβ from the brain in an animal model, which was assumed tobe due to an enhanced transport of Aβ carrier proteins such as albuminand transthyretin into the brain (Carro et al., Nat Med 2002; 8:1390-7).

A typical protocol for amyloid dialysis according to any of theembodiments above would comprise selecting a patient who is at risk foror suffers from Alzheimer's Disease, and purifying in vivo or ex vivobodily fluids of the patient from free Aβ. Specific protocols using theexemplary Aβ-ligand apoE are provided in the Examples. Typically, thedialysis is conducted daily or weekly for as long as necessary for: (i)at least one relevant symptom of Alzheimer's to abate; or (ii) for thelevel of Aβ peptide in the blood to be reduced or to be significantlyundetectable, or some combination thereof.

The efficacy of the treatment can be determined by evaluating the ADsymptoms of the patient and/or by measuring the Aβ concentration in thepatient's blood. Blood measurements typically involve taking a bloodsample, separating serum from red blood cells, and usingradioimmunoassay, enzyme linked immunosorbent assay (ELISA) orchromatographic analysis of the Aβ peptide contents in serum.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

The term “hemofiltration” refers to a process of filtering blood by amembrane resulting in the separation of all proteins larger than theeffective pore size of the membrane from retained plasma water andsolute (these return to the patient) from the ultrafiltrate.

The term “hemofilter” refers to the filter used in hemofiltration. Itcan be configured in a number of ways, e.g., as a series of parallelplates or as a bundle of hollow fibers. The blood path is from a bloodinlet port, through the fibers or between the plates, then on to a bloodoutlet port. Filtration of blood occurs at the membrane withultrafiltrate forming on the side of the membrane opposite the blood.This ultrafiltrate accumulates inside the body of the filter containedand embodied by the filter jacket. This jacket has an ultrafiltratedrainage port.

The term “ultrafiltrate” refers to the filtered plasma water and soluteand molecules smaller than the effective pore size of the membrane.

The subject or patient to which the present invention may be applicablecan be any vertebrate species, preferably mammalian, including but notlimited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats,cats, dogs, hamsters, mice, rats, rabbits, monkeys, chimpanzees, andhumans. In a preferred embodiment, the subject is a human. The inventionis particularly applicable for human subjects at risk for or sufferingfrom Alzheimer's Disease (AD).

A “control” is an alternative subject or sample used in an experimentfor comparison purposes. A control can be “positive” or “negative”. Forexample, where the purpose of the experiment or the comparison in amethod is to determine a correlation of an patient treatment with aparticular symptom, one may use either a positive control (a patientexhibiting the symptom and not subjected to the treatment, or a samplefrom such a patient), and/or a negative control (a healthy subject notsubjected to the treatment).

As used herein, “treatment” Generally refers to a method to reduce theconcentration or amount of Aβ in the blood or CVF compartment,including, but not limited to, dialysis or plasma exchange, or theadministration of protein or peptides capable of associating in vivowith Aβ in bodily fluids. “Treatment” also includes prophylactictreatment to those at risk for amyloid diseases, i.e., familialAlzheimer's disease.

The term “bodily fluid” as used herein includes blood, plasma, serum,cerebroventricular fluid, cerebrospinal fluid, and other extracellularor interstitial fluids in the body of a subject.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range oftip to 20%, preferably tip to 10%, more preferably tip to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g. Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription and Translation (B. D. Hames & S. J. Higgins,eds. (1984)): Animal Cell Culture (R. I. Freshney, ed. (1986)):Immobilized Cell and Enzymes (IRL Press, (1986)); B. Perbal. A practicalGuide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Molecular Biology

A “nucleic acid molecule” refers to the phosphate ester polymeric formof ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNAmolecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine,deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoesteranalogs thereof, such as phosphorothioates and thioesters, in eithersingle stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includesdouble-stranded DNA found inter alia, in linear (e.g., restrictionfragments) or circular DNA molecules, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

A polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in a nucleic acid, such as DNA andRNA, and means any chain of two or more nucleotides. A nucleotidesequence typically carries genetic information, including theinformation used by cellular machinery to make proteins and enzymes.These terms include double or single stranded genomic and cDNA, RNA, anysynthetic and genetically manipulated polynucleotide, and both sense andanti-sense polynucleotide (although only sense strands are beingrepresented herein). This includes single- and double-strandedmolecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as“protein nucleic acids” (PNA) formed by conjugating bases to an aminoacid backbone. This also includes nucleic acids containing modifiedbases, for example thio-uracile, thio-guanine and fluoro-uracil.

The nucleic acids herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters,phosphoroamidates, carbamates, etc.) and with charged linkages (e.g.,phosphorothioates, phosphorodithioates, etc.). Polynucleotides maycontain one or more additional covalently linked moieties, such as, forexample, proteins (e.g., nucleases, toxins, antibodies, signal peptides,poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.),chelators (e.g., metals, radioactive metals, iron, oxidative metals,etc.), and alkylators. The polynucleotides may be derivatized byformation of a methyl or ethyl phosphotriester or an alkylphosphoramidate linkage. Furthermore, the polynucleotides herein mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for die binding of RNApolymerase. The promoter may be operatively associated with otherexpression control sequences, including enhancer and repressorsequences.

Promoters which may be used to control gene expression include, but arenot limited to, cytomegalovirus (CM V) promoter (U.S. Pat. No. 5,385,839and No. 5,168,062), the SV40 early promoter region (Benoist and Chambon,Nature 1981; 290:304-310), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 1980;22:787-797), the herpes thymidine kinase promoter (Wagner et al., Proc.Natl. Acad. Sci. USA 1981; 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., Nature 1982; 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Komaroff, et al., Proc. Natl. Acad. Sci. USA 1978; 75:3727-3731),or the lac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. USA 1983;80:21-25); see also “Useful proteins from recombinant bacteria” inScientific American 1980, 242:74-94; promoter elements from yeast orother fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase)promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatasepromoter, and transcriptional control regions that exhibit hematopoietictissue specificity, in particular: beta-globin gene control region whichis active in myeloid cells (Mogram et al., Nature 1985; 315:338-340;Kollias et al., Cell 1986; 46:89-94), hematopoietic stem celldifferentiation factor promoters, erythropoietin receptor promoter(Maouche et al., Blood 1991; 15:2557), etc.

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

A coding sequence is “under the control of” or “operatively associatedwith” transcriptional and translational control sequences in a cell whenRNA polymerase transcribes the coding sequence into RNA, particularlymRNA, which is then trans-RNA spliced (if it contains introns) andtranslated into the protein encoded by the coding sequence.

As used herein, the term “isolated” means that the referenced materialis removed from its native environment, e.g., a cell. Thus, an isolatedbiological material can be free of some or all cellular components,i.e., components of the cells in which the native material is occursnaturally (e.g., cytoplasmic or membrane component). A material shall bedeemed isolated if it is present in a cell extract or if it is presentin a heterologous cell or cell extract. In the case of nucleic acidmolecules, an isolated nucleic acid includes a PCR product, an isolatedmRNA, a cDNA, or a restriction fragment. In another embodiment, anisolated nucleic acid is preferably excised from the chromosome in whichit may be found, and more preferably is no longer joined or proximal tonon-coding regions (but may be joined to its native regulatory regionsor portions thereof, or to other genes, located upstream or downstreamof the gene contained by the isolated nucleic acid molecule when foundin the chromosome. In yet another embodiment, the isolated nucleic acidlacks one or more introns. Isolated nucleic acid molecules includesequences inserted into plasmids, cosmids, artificial chromosomes, andthe like, i.e., when it forms part of a chimeric recombinant nucleicacid construct. Thus, in a specific embodiment, a recombinant nucleicacid is an isolated nucleic acid. An isolated protein may be associatedwith other proteins or nucleic acids, or both, with which it associatesin the cell, or with cellular membranes if it is a membrane-associatedprotein. An isolated organelle, cell, or tissue is removed from theanatomical site in which it is found in an organism. An isolatedmaterial may be, but need not be, purified.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. As used herein,the term “substantially free” is used operationally, in the context ofanalytical testing of the material. Preferably, purified materialsubstantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

Methods for purification are well-known in the art. For example, nucleicacids can be purified by precipitation, chromatography (includingwithout limitation preparative solid phase chromatography,oligonucleotide hybridization, and triple helix chromatography),ultracentrifugation, and other means. Polypeptides and proteins can bepurified by various methods including, without limitation, preparativedisc-gel electrophoresis and isoelectric focusing; affinity, HPLC,reversed-phase HPLC, gel filtration or size exclusion, ion exchange andpartition chromatography; precipitation and salting-out chromatography;extraction; and countercurrent distribution. For some purposes, it ispreferable to produce the polypeptide in a recombinant system in whichthe protein contains an additional sequence tag that facilitatespurification, such as, but not limited to, a polyhistidine sequence, ora sequence that specifically binds to an antibody, such as FLAG and GST.The polypeptide can then be purified from a crude lysate of the hostcell by chromatography on an appropriate solid-phase matrix.Alternatively, antibodies produced against the protein or againstpeptides derived therefrom can be used as purification reagents. Cellscan be purified by various techniques, including centrifugation, matrixseparation (e.g., nylon wool separation), panning and otherimmunoselection techniques, depletion (e.g., complement depletion ofcontaminating cells), and cell sorting (e.g., fluorescence activatedcell sorting (FACS)). Other purification methods are possible andcontemplated herein. A purified material may contain less than about50%, preferably less than about 75%, and most preferably less than about90) %, of the cellular components, media, proteins, or othernondesirable components or impurities (as context requires), with whichit was originally associated. The term “substantially pure” indicatesthe highest degree of purity which can be achieved using conventionalpurification techniques known in the art.

The term “express” and “expression” means allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing RNA (such as rRNA or mRNA) or a protein by activating thecellular functions involved in transcription and translation of acorresponding gene or DNA sequence. A DNA sequence is expressed by acell to form an “expression product” such as an RNA (e.g. a mRNA or arRNA) or a protein. The expression product itself, e.g., the resultingRNA or protein, may also said to be “expressed” by the cell.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include E. coli host cells and plasmidvectors, insect host cells such as Sf9, Hi5 or S2 cells and Baculovirusvectors, Drosophila cells (Schneider cells) and expression systems, fishcells and expression systems (including, for example, RTH-149 cells fromrainbow trout, which are available from the American Type CultureCollection and have been assigned the accession no. CRL-1710) andmammalian host cells and vectors.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e., extrinsic or extracellular) gene, DNA or RNA sequenceinto a host cell so that the host cell will express the introduced geneor sequence to produce a desired substance, in this invention typicallyan RNA coded by the introduced gene or sequence, but also a protein oran enzyme coded by the introduced gene or sequence. The introduced geneor sequence may also be called a “cloned” or “foreign” gene or sequence,may include regulatory or control sequences (e.g., start, stop,promoter, signal, secretion or other sequences used by a cell's geneticmachinery). The gene or sequence may include nonfunctional sequences orsequences with no known function. A host cell that receives andexpresses introduced DNA or RNA has been “transformed” and is a“transformant” or a “clone”. The DNA or RNA introduced to a host cellcan come from any source, including cells of the same genus or speciesas the host cell or cells of a different genus or species.

The terms “vector”, “cloning vector” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors may include plasmids, phages, viruses, etc. and arediscussed in greater detail below.

Amyloid-Beta (Aβ) Peptides

Aβ is a 4.1-4.3 kD hydrophobic peptide that is encoded on chromosome 21as part of a much longer amyloid precursor protein APP (Muller-Hill etal., Nucleic Acids Res 1989; 17:517-522). The APP protein starts with aleader sequence (signal peptide), followed by a cysteine-rich region, anacidic-rich domain, a protease inhibitor motif, a putativeN-glycosylated region, a transmembrane domain, and finally a smallcytoplasmic region. The Aβ sequence begins close to the membrane on theextracellular side and ends within the membrane. Two-thirds of Aβ facesthe extracellular space, and the other third is embedded in the membrane(Kang et al., Nature 1984; 325:733-736, 1987 and Dyrks et al., EMBO J1988, 7:949-957). Several lines of evidence suggest that amyloid mayplay a central role in the early pathogenesis of AD (Soto et al., 1994;63:1191-1198).

The present invention provides methods of treating an individualsuffering from an amyloid disease by removing Aβ present in theindividuals bloodstream or other bodily fluid. According to oneembodiment of the invention, compounds associated with Aβ (hereinafteralternatively referred to as “binding compounds”), or fragments of suchcompounds, are administered to a patient suffering from or at risk foran amyloid disease. Such binding compounds are described below.

Aβ Binding Compounds

Aβ “binding compounds” or Aβ “ligands” herein are molecules that bind toAβ, including Aβ1-40 and Aβ1-42. Exemplary ligands are listed in Table1, and also include monoclonal antibodies or fragments thereof,synthetic ligands, and the like, and which specifically bind Aβ.

Non-limiting examples of the Aβ binding compounds or ligands for use inpresent invention are apolipoprotein E, apolipoprotein J, serum amyloidP component, RNA aptamers directed against Aβ, α1-antichymotrypsin,proteoglycans, gangliosides (such as monosiologanglioside GM1),vitronectin, vimentin, and combinations thereof. These are shown inTable 1 below along with commercial sources for the ligands.

TABLE 1 Amyloid-β binding compound Company Source Serum Amyloid PBiogenesis Human serum α 1-antichymotrypsin Biodesign, USBio, Humanplasma Biogenesis, ICN, Cortex, Scipac Apolipoprotein E ICN, USBio,Biodesign, Human plasma, Filzgerald, Biogenesis, human Cortexrecombinant Vitronectin Calbiochem, Chemicon Human plasma Promega,Sigma, Apolipoprotein E4 ICN Human recombinant Apolipoprotein E3 ICNHuman recombinant Apolipoprotein E2 Biogenesis Human recombinantApolipoprotein J Quidel Human, purified Heparan Sulfate Sigma Mousesarcoma Proteoglycan Monosiologanglioside GM1 Sigma Bovine brainMonosiologanglioside GM2 Sigma Bovine brain Monosiologanglioside GM3Sigma Bovine brain Disialoganglioside GD1a Sigma Bovine brainDisialoganglioside GD1b Sigma Bovine brain Trisialoganglioside GT1bSigma Bovine brain Gangliosides mixture Sigma Bovine brain VimentinSigma, Biodesign, Bovine lens Biogenesis Human serum albumin ICN, Sigma,Biogenesis Human serum Human transthyretin Biogenesis, Sigma Humanplasma

In addition to these sources, the compounds listed in Table 1 can bepurified from human plasma and/or various human tissues. Moreover,proteinaceous compounds can be produced recombinantly using expressionsystems known to those of ordinary skill in the art disclosed above andthe DNA sequences set forth in public databases (World-Wide Web atncbi.nlm.nih.gov/LocuLink/). The recombinant proteins can be purifiedusing standard techniques well known to those of ordinary skill in theart disclosed above.

One or more of the compounds may also be conjugated to a second moiety.The moiety can be a molecule that prevents degradation and/or increaseshalf-life, reduces toxicity, reduces immunogenicity, facilitatestransport over the blood-brain-barrier, or increases biological activityof the Aβ ligand. Exemplary vehicles include human serum albumin or anynatural or synthetic protein or polypeptide, an Fc domain (see, e.g.,U.S. Pat. No. 6,660,843); a linear polymer (e.g., polyethylene glycol(PEG), polylysine, dextran, etc.); a branched-chain polymer (see, forexample, U.S. Pat. Nos. 4,289,872 and 5,229,490 or PCT publication WO93/21259); a lipid; a cholesterol group (such as a steroid); acarbohydrate or oligosaccharide. The ligand may be conjugated to thesecond moiety directly or via a linker. See U.S. Pat. No. 6,660,843 forgeneral descriptions of useful conjugation techniques.

Those of ordinary skill in the art will also appreciate that mimetics ofthese Aβ binding compounds can be used. For example, when the compoundhas a peptide backbone, the peptide bonds can be replaced withnon-peptide bonds. Peptidomimetics can have various different structures(Ripka et al., Curr. Opin. Chem. Biol. 1998; 2:441-452). For example,peptidomimetics can be: (I) peptide analogues containing one or moreamide bond replacements (Spatola, A. F., In Chem. Biochem. Amino Acids,Pept., Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1983; pp267-257); (2) peptide analogues with various conformational restraints(Hart, P. A.; Rich, D. H., In Pract. Med. Chem.; Wermuth, C., Ed.; Acad.Press: London, U.K., 1996; pp 393-412), (3) novel structures thatreplace the entire peptide backbone while retaining isosteric topographyof the peptide (Farmer, P. S., In Drug Design, Ariens, E. J., Ed.;Academic Press: New York, 1980; Vol. 10, pp 119-143), and (4) variousheterocyclic natural products or screening leads that mimic the functionof the natural peptide (Fletcher, M. D. and Campbell, M. M., Chem. Rev.1998; 98:763-795).

RNA aptamers directed against Aβ can be used to treat amyloid diseasepursuant to the present invention. RNA aptamers directed against Aβ arehigh affinity ligands selected from a combinatorial library described inYlera et al. (Biochem. Biophys. Res. Comm. 2002; 200:1583-1588). SuchRNA aptamers can be isolated as disclosed in Ylera et al. (supra) or canbe chemically synthesized since Ylera et al. provide the nucleotidesequence of a number of Aβ specific RNA aptamers.

The term “fragment” as used herein refers to the compounds of thepresent invention which are peptides containing less than the full aminoacid sequence of the active parent protein but retain their bindingactivity to Aβ. Fragments or their mimetics can be detected by standardELISA-based binding assays (Tokuda et al., Biochem J. 2000; 348:359-65);phage display techniques (Rodi et al., Curr Opin Biotechnol 1999;10(1):87-93); yeast two hybrid systems (Uetz P., Curr Opin Chem Biol2002; 6(1):57-62), and/or protein microarray technology (Templin et al.,Trends Biotechinol 2002; 20(4):160-6). These assays can also be used toscreen for novel Aβ binding compounds.

Antibodies

In an alternative embodiment, the Aβ ligand or Aβ binding compound is anantibody. The antibodies useful herein can be polyclonal or monoclonal,native or engineered in any suitable manner, and are selective for theparticular target molecule. Such antibodies are conveniently made usingthe methods and compositions disclosed in Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring, Harbor Laboratory, 1988, as well asimmunological and hybridoma technologies known to those skilled in theart.

The term “antibody” is intended to include immunoglobulins of allisotypes and species. The antibody can be of can be any type, e.g., anIgG, IgE, IgM, IgD or IgA, preferably, the antibody is an IgG. Inanother specific embodiment, the construct is derived from aT-lymphocyte receptor. Additionally, the antibody may be of any subclassor isotype of each particular class of antibodies.

A “monoclonal antibody” is an immunoglobulin secreted by a single cloneof cells. Any technique that provides for the production of antibodymolecules by continuous cell lines in culture may be used. These includebut are not limited to the hybridoma technique originally developed byKöhler and Milstein (Nature 1975; 256:495-497), as well as the triomatechnique, the human B cell hybridoma technique (Kozbor et al.,Immunology Today 1983; 4:72 et seq.; Cote et al., Proc. Natl. Acad. Sci.USA 1983; 80:2026-2030), and the EBV hybridoma technique to producehuman monoclonal antibodies (Cole et al., in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). In an additionalembodiment of the invention, monoclonal antibodies can be produced ingerm free animals (see International PCT Publication WO 89/12690).Particular isotypes of monoclonal antibodies can be prepared eitherdirectly by selecting from the initial fusion, or prepared secondarily,from a parental hybridoma secreting a monoclonal antibody of differentisotype by using the sib selection technique to isolate class switchvariants (Steplewski et al., Proc. Natl. Acad. Sci. USA 1985; 82:8653 etseq.; Spira et al., J. Immunol. Meth. 1984; 74:307 et seq.).

Fragments of an immunoglobulin family protein that are specific to atarget molecule can also be prepared. For example, such fragmentsinclude but are not limited to: F(ab′)₂ fragments that contain thevariable regions of both the heavy and the light chains, the lightconstant region and the CH1 domain of the heavy chain, which fragmentscan be generated by pepsin digestion of all antibody; Fab′ fragments;Fab fragments generated by reducing the disulfide bonds of an F(ab′)₂fragment (King et al., Biochem. J. 1992; 281:317 et seq.); and Fvfragments, i.e., fragments that contain the variable region domains ofboth the heavy and light chains (Reichmann and Winter, J. Mol. Biol.1988; 203:825 et seq.; King et al., Biochem J. 1993; 290:723 et seq.).

Single chain antibodies (SCA) may also be prepared (U.S. Pat. No.4,946,778; Bird, Science 1988; 242:423-426; Huston et al., Proc. Natl.Acad. Sci. USA 1988, 85:5879-5883; and Ward et al., Nature 1989;334:544-546). Single chain antibodies are formed by linking the heavyand light chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Additionally, the inventionalso provides heavy chain and light chain dimers and diabodies.

Modified chimeric or humanized antibodies may also be prepared. Achimeric antibody is a molecule in which different portions of theantibody molecule are derived from different animal species, such asthose having a variable region derived from a murine monoclonal antibodyand a constant region derived from a human immunoglobulin constantregion. Techniques have been developed for the production of chimericantibodies (Morrison et al., Proc. Natl. Acad. Sci. USA 1984;81:6851-6855; Neuberger et al., Nature 1984; 312:604-608; Takeda et al.,Nature 1985; 314:452-454; Oi et al., BioTechniques, 1986; 4:214 et seq.;and International Patent Application No. PCT/GB85/00392) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity. In a specific embodiment, the chimeric antibodycontains the variable domain of a non-human antibody and the constantdomain of a human antibody. In another embodiment, the construct isderived from a humanized antibody, in which the CDRs of the antibody(except for the one or more CDRs containing the heterologous bindingsequence) are derived from an antibody of a non human animal and theframework regions and constant region are from a human antibody (see,U.S. Pat. No. 5,225,539; and Oi et al., supra). The creation ofcompletely human monoclonal antibodies is possible through the use oftransgenic mice in which the mouse immunoglobulin gene loci have beenreplaced with human immunoglobulin loci to provide in vivoaffinity-maturation machinery for the production of humanimmunoglobulins.

Formulation

The present invention also provides methods of using pharmaceuticalcompositions of the Aβ ligands described herein. Such pharmaceuticalcompositions may be for administration for injection, or for oral,pulmonary, nasal, transdermal or other forms of administration. Ingeneral, the invention encompasses pharmaceutical compositionscomprising effective amounts of an Aβ ligands together withpharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents of various buffer content (e.g., Tris-HCl, acetate, phosphate),pH and ionic strength; additives such as detergents and solubilizingagents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol); incorporationof the material into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc. or into liposomes.Hyaluronic acid may also be used, and this may have the effect ofpromoting sustained duration in the circulation. Such compositions mayinfluence the physical state, stability, rate of in vivo release, andrate or in vivo clearance of the present proteins and derivatives. See,e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, Pa. 18042) pages 1435-1712 which are hereinincorporated by reference. The compositions may be prepared in liquidform, or may be in dried powder, such as lyophilized form. Implantablesustained release formulations are also contemplated, as are transdermalformulations.

Also contemplated herein is pulmonary delivery of the present Aβligands. The Aβ ligand is delivered to the lungs of a mammal whileinhaling and traverses across the lung epithelial lining to the bloodstream. (Other reports of this include Adjei et al., Pharma. Res. 1990;7:565-9; Adjei et al., Internatl. J. Pharmaceutics 1990; 63:135-44(leuprolide acetate); Braquet et al., J. Cardiovasc. Pharmacol. 1989;13(suppl. 5):s.143-146 (endothelin-1); Hubbard et al., Annals Int. Med.1989; 3:206-12 (.alpha.1-antitrypsin); Smith et al., J. Clin. Invest.1989; 84:1145-6 (.alpha. 1-proteinase); Oswein et al. (March 1990),“Aerosolization of Proteins”, Proc. Symp. Resp. Drug Delivery II,Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988),J. Immunol. 140: 3482-8 (interferon-65 and tumor necrosis factor alpha.)and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colonystimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C.; and the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass. All such devices requirethe use of formulations suitable for the dispensing of the Aβ ligand.Typically, each formulation is specific to the type of device employedand may involve the use of an appropriate propellant material, inaddition to diluents, adjuvants and/or carriers useful in therapy. Theinventive compound should most advantageously be prepared in particulateform with an average particle size of less than 10 .mu.m (or microns),most preferably 0.5 to 5 μm, for most effective delivery to the distallung.

Pharmaceutically acceptable carriers include carbohydrates such astrehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Otheringredients for use in formulations may include DPPC, DOPE, DSPC andDOPC. Natural or synthetic surfactants may be used. PEG may be used(even apart from its use in derivatizing the protein or analog).Dextrans, such as cyclodextran, may be used. Bile salts and otherrelated enhancers may be used. Cellulose and cellulose derivatives maybe used. Amino acids may be used, such as use in a buffer formulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers known in the art, is contemplated.

In Vivo Treatment

Treatment in vivo, i.e., by a method where an Aβ-binding compound isadministered to the patient, is expected to result in reduced amyloidburden within the brain of an Alzheimer's patient and has the potentialto halt or slow the progression of the cognitive impairments observed inthe disease. In other amyloid diseases, this treatment approach isexpected to enhance clearance of the respective amyloid proteins fromtheir target organs in a similar manner and therefore improve thecondition of those patients.

Following administration, the compound will bind to Aβ. The Aβ ispreferably, although not necessarily, soluble Aβ. The Aβ is free, e.g,not irreversibly bound to an amyloid plaque or other component. Free Aβincludes, but is not limited to, circulating Aβ in blood, free Aβ in theinterstitial fluid in the brain; and Aβ bound to a ligand such as anaturally occurring plasma protein, e.g., albumin or transthyretin. Inthis embodiment, the compound preferably has a higher affinity to Aβthan the plasma protein, so that Aβ preferentially binds theadministered compound. Normally, equilibrium is presumed to existbetween free Aβ in circulation and Aβ within the brain or other affectedorgans. A reduction in free Aβ in the circulation by administration ofan Aβ ligand which does not cross the blood-brain-barrier can thereforeresult in an efflux of Aβ out of the brain or other similarly affectedorgans to re-establish the equilibrium. The bound Aβ will be broken downin the liver and excreted. The subsequent reduction in Aβ within, e.g.,the brain leaves less Aβ available for aggregation/fibril formation.

In one embodiment, the patient is treated in a manner so as to increasethe selective permeability of the blood-brain barrier, allowing thetransport of a particular Aβ ligand into the brain from the blood. Inthis embodiment, the level of Aβ in the brain can be reduced as theAβ-ligand complex is transported back into the blood circulation.Treatments to selectively increase the permeability of theblood-brain-barrier to certain Aβ binding compounds in a patientinclude, but are not limited to, the administration of about 1 to about1000 μg/kg body weight, preferably about 10 to about 100 μg/kgbodyweight, of IGF-I as a bolus injection to a patient about 0.5 to 10hours, preferably about 1 hour, before administration of Aβ bindingcompounds. While not being bound to any specific theory, this treatmentcan enhance selective endocytosis of large molecules such as Aβ bindingproteins (Carro et al., Nature Med 2002; 8:1390-1397). Also, evenwithout selective permeabilization with a drug such as IGF-I, the bloodbrain barrier may be compromised in Alzhemier's disease so thatAβ-binding compounds that normally do not enter the brain, or have asaturated uptake, may access the brain more readily. Hence, Aβ clearancemediated by these compounds may be partially from within the brain. Aβbound to its binding compound or carrier may be shuttled out of thebrain or be degraded within the central nervous system. The net effectwill be a reduced concentration of Aβ within the interstitial fluid.

The compounds of the present invention may be administered systemically.The term “systemic” as used herein includes parenteral, topical, oral,spray inhalation, rectal, nasal and bucal administration. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, and intraperitoneal administration. Preferably, thecompositions are administered orally or intravenously in effectiveamounts to treat the amyloid diseases.

The specific dosage regimen and amounts administered involved in amethod for treating the above-described conditions will be determined bythe attending physician, considering various factors which modify theaction of drugs, e.g. the age, condition, body weight, sex and diet ofthe patient, the severity of any infection, time of administration andother clinical factors. An effective amount to treat the diseases wouldbroadly range between about 0.1 mg and about 10 mg per kg body weight ofthe recipient per day and may be administered as a single or divideddoses. Specifically, the amount administered would range between aboutone-tenth and up to about two-fold of mean plasma levels of the Aβbinding compound. The amount administered of any of the compounds wouldbe no greater than needed to bind all free Aβ in plasma. The treatmentscan be continued throughout the life of the patient.

The approximate naturally occurring plasma concentrations of Aβ bindingcompounds and soluble Aβ (sAβ) are as follows:

TABLE 2 Proteins/Peptides Mean Plasma Levels (μg/ml) Apolipoprotein E(monomer) 54 Apolipoprotein E (dimer) 54 Apolipoprotein J 100α1-antichymotrypsin 40 serum amyloid P component 34 sAβ .001

Dialysis

In an alternative embodiment, a reduction in free Aβ is achieved not byadministering the compounds to the patient but by dialyzing a patient'sblood through a column and/or membrane to remove the Aβ protein from thepatient's blood. The column or membrane may contain the amyloid-bindingcompounds of the present invention covalently attached thereto. Usingthis approach, the patient will not be directly exposed to theseendogenous compounds. The reduction of free Aβ as a result of dialysiswill result in an efflux of Aβ out of the brain or similarly affectedorgans in order to re-establish the equilibrium. The subsequentreduction in free Aβ by virtue of dialyzation leaves less Aβ availablefor aggregation/fibril formation. Similar to the above described in vivotreatment method, this ex vivo treatment method is expected to result inreduced amyloid burden within the brain of an Alzheimer's patient andhas the potential to hall or slow the progression of the cognitiveimpairments observed in the disease.

The dialyzing blood treatments of the invention can be used to reduce oreliminate the presence of specific Aβ peptides free flowing in plasma.Dialysis eliminates the concerns over adverse immune response or otheradverse responses to synthetic constructs or monoclonal antibodiesbecause such constructs or monoclonal antibodies are not introduced intothe patient's body. Furthermore, dialysis allows instant initiation andcessation of treatment. Preferred methods of dialysis which may be usedin the present invention include, but are not limited to, hemodialysis,plasma exchange, plasma perfusion, and hemofiltration. The latter threetechnologies do not require Aβ binding compounds. The methods may beconducted on a continuous or batch basis. Treated blood may be returnedto the patient concurrently with treatment or following treatment. Theblood may be supplemented or reconstituted with components from donatedblood, artificial or synthetic components.

Hemodialysis is the most common method used to treat advanced andpermanent kidney failure. It consists of two compartments separated by asemi-permeable membrane. One compartment is filled with blood, the otheris filled with a solution of certain minerals and water (referred to asthe dialysate bath). Normal blood is 90% water. Water molecules willpass through the membrane freely back and forth. Blood also containswhite and red blood cells, protein, fat, sugar, minerals and wasteproducts. The red and white blood cells are too large to pass throughthe membrane so they remain in the blood compartment. The same is trueof fat and protein molecules. However, electrolytes, because of theirsmaller size, pass freely through the membrane in both directions(principle of diffusion). This principle states that particles in asolution of high concentration pass through a semi-permeable membraneinto a solution of lower concentration until there is an equalconcentration of particles on both sides (concentration gradient). Theconcentration of electrolytes is adjusted in the bath side toapproximate the levels in normal human blood serum. Metabolic wasteproducts (urea, creatinine etc.) in the blood (larger molecules butsmall enough to pass through the membrane) are removed utilizing theprinciple of diffusion. When the concentration of the waste productsreach the levels of the blood, the bath solution is changed eitherperiodically or continuously.

For use in the present invention, Aβ binding compound are added to thedialysis bath. The semi-permeable membrane will have a molecular weightcutoff at 10,000 Daltons. Soluble free Aβ monomers and dimers in theblood will diffuse into the dialysis bath and bind to the Aβ bindingcompounds. Thereafter, Aβ will not diffuse back into the blood.

The Aβ binding compound in the dialysis compartment may have a high,moderate, or relatively low affinity for Aβ. Compounds having relativelylow affinity include albumin and transthyretin. The Aβ-ligandconcentration and incubation time in the dialysis compartment can beoptimized for each Aβ-ligand, taking affinity and other relevantphysicochemical properties into consideration.

In hemofiltration procedures, the principle used to eliminate the wasteproducts is different. Solute (in most cases the blood) is carriedacross a semi-permeable membrane in response to a transmembrane pressuregradient (a process known as solvent drag). This mimics what actuallyhappens in the normal human kidney. The rate of the ultrafiltrationdepends upon blood now. This is very effective in removal of fluid andmiddle sized molecules, which are thought to cause uremia.

When this method is used, there is no need for Aβ binding compounds. Themembranes that are normally used in this technique allow the passage ofmolecules with a molecular weight of less than 20,000 Daltons.Filtration across the membrane is convective, which means that it isunidirectional. Therefore, filtered Aβ cannot flow back.

Typical procedures also include plasma perfusion (also known as plasmaexchange or plasmapheresis): Plasma is the fluid portion of the bloodthat allows circulation of red blood cells, white blood cells andplatelets. It consists of mainly water and numerous chemical compounds.Plasma exchange involves the separation and removal of the plasma fromthe blood in order to remove disease substances circulating in theplasma. The red and white blood cells and platelets are returned to thepatient, along with a replacement fluid. Plasma exchange is accomplishedwith a device called a blood cell separator. Centrifuge or membranefilters are used to separate plasma from cellular blood components.Blood is drawn from a patient's arm vein by a needle which is attachedto a blood tubing set. After it goes through the blood cell separator,the cellular components are drawn from the compartment and replacementfluid prescribed by the physician is added to the cellular components.The mix is returned to the patient usually through a needle. All thesteps mentioned above can be done in an automated, continuous and safemanner.

When plasma perfusion is used pursuant to the present invention, Aβbinding compounds are not needed. This approach involves removing theplasma from the patient, while the blood cells and platelets arereturned to the patient with replacement fluid.

A typical protocol for dialysis would comprise selecting patients whohave Down's syndrome, mild cognitive impairment or those at risk forAlzheimer's disease and conducting dialysis using as a binding memberthe compounds or fragments thereof associated with Aβ as defined above.Preferably, the dialysis takes place over a period of 2-3 hours, and isrepeated as necessary. Typically, dialysis is conducted every 1-7 daysfor as long as the concentration of free Aβ in the patient's bloodremains high, e.g., above 0.1-0.5 ng/ml (10-50% of mean plasma level).

The efficacy of the treatment can be evaluated by either evaluating thesymptoms of the patient or by measuring the concentration or amount oftarget molecules (Aβ) in the patient's blood. The amount of Aβ in thepatient's blood can be determined by enzyme linked immunosorbent assay(ELISA) as described below.

In another preferred embodiment of the present invention, the Aβ bindingcompounds are immobilized, i.e., fixed so that neither the bindingcompounds nor the binding pair travel with the blood. Preferably, thebinding partner construct is immobilized on a solid support usingcovalent or affinity binding. Covalent linkage can be achieved usingstandard cyanogen bromide (CNBr) or other activation techniques(International PCT publication WO 00/74824 by Bristow, European PatentNo. 272 792 to Jones, U.S. Pat. No. 5,122,112 to Jones), or a highaffinity interaction, such as that between avidin and biotin(International PCT publication WO 00/74824 by Bristow; U.S. Pat. No.6,251,394 to Nilsson). An antibody binding compound can be attached viaits Fc region, if present, to a protein-A column (Kiprov is et al., J.Biol. Res. Mod. 1984; 3:341-346; Jones et al., J. Biol. Res. Mod. 1984;3:286-292; Besa et al., Am. J. Med. 1981; 71:1035-1040; EP Application172018 of Bensinger; EP Application 079221 of Terman; and U.S. Pat. No.4,614,513 to Bensinger).

To regenerate the solid support containing the binding compounds afteruse, bound Aβ may be removed preferably by altering the pH and/or by theuse of chaotrophic agents. Since the binding compound is notadministered to the patient in this embodiment of the present invention,the binding compound may be an antibody. Such antibodies arecommercially available from numerous sources such as Bachem, Biogenesis,Biosource, Calbiochem, Chemicon and Sigma. When the binding partner isan antibody, it may be attached via its Fc region to a solid support ormembrane.

The solid support utilized in dialysis devices and methods can be madeout of a variety of substances (nitrocellulose, cellulose, nylon,plastic, rubber, polyacrylamide, agarose,poly(vinylalcoholo-co-ethylene), and can be formed in a variety ofshapes, including flat dialyzers, semi-permeable membranes,semi-permeable hollow fibers, coils, permeable spheres, dialysismembranes, and plasmapheresis filters, optionally using linker moleculessuch as PEG (polyethelene glycol) to attach the ligand (as disclosed inWO 00/74824). In a hemofiltration device, the solid supports may be, forexample, beads, plates, hollow filters, or any combination thereof. Oneparticular method which can be used in the present invention is designedto remove small, non-protein-bound toxins using hollow-fiber technologyas disclosed in U.S. Pat. No. 5,919,369.

In ex vivo dialysis procedures, the binding compounds described hereincan be used in amounts sufficient to remove the target molecule (Aβ)completely from the blood or simply to reduce the amount of the moleculein the blood. The precise amount of the constructs to be employeddepends on the efficiency of the apparatus used and the expected amountsof target molecule in the blood. The amount of binding partner to beimmobilized on the solid support can also vary depending oil theaffinity between binding, partner and target, type of perfusion device,and length of perfusion treatment. These amounts can be determinedaccording to the judgment of the practitioner and each patient'scircumstances according to standard clinical techniques. Typically,however, the amount of immobilized binding partner would range betweenabout 50- and about 1000-fold molar excess compared to free Aβ in theblood.

Various designs for hemodialysis and dialysis devices are known and havebeen described in, e.g., patent literature (see, e.g., U.S. Pat. Nos.4,824,432; 5,122,112; 5,919,369; and 6,287,510; and PCT applicationspublished as WO 90/15631; WO 00/74824; WO 01/37900; and WO 01/45769).

In addition, patients suffering from amyloid disease can be treated by acombination of methods, i.e., administration of the compounds associatedwith Aβ of the present invention and dialysis. For example, patients caninitially be treated using dialysis to rapidly remove the circulating,free Aβ until the amount is stabilized at about 10-50% of the normalvalue. Thereafter, the compounds of the present invention can beadministered, thereby minimizing the number of invasive dialysistreatments

The present invention will be better understood by reference to thefollowing examples, which are provided as exemplary of the invention,and not to limit the scope thereof.

EXAMPLES Example 1 Binding of ApoE Preparations to Aβ Peptide

In the present Example, the binding of apoE, derived from varioussources and in various forms, to Aβ1-40 and Aβ1-42 peptides isevaluated.

Materials and Methods

Synthetic Peptides and Proteins. The following peptides,

(SEQ ID NO:1) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV, and (SEQ IDNO:2) DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA

corresponding to Aβ40 and Aβ42, and identical to residues 672-711 and672-713 of Aβ-precursor protein 770, respectively, were synthesized atthe W. M. Keck Facility at Yale University (New Haven, Conn., U.S.A.)using N-t-butyloxycarbonyl chemistry and purified by HPLC. Aliquots ofthe final products were lyophilized and stored at −20° C. until use. Forpreparation of aggregated peptides, 50 μg of either Aβ340 or Aβ42 weredissolved in 100 μl of PBS (0.02 M phosphate buffer, pH 7.4, containing0.15 M NaCl) and incubated at 37° C. for 72 h. ApoE3 and apoE4 producedin Sf9 insect cells by the baculovirus expression system were purchasedfrom PanVera (Madison, Wis., U.S.A.). In all cases, protein purity wascorroborated by SDS/PAGE and N-terminal sequence analysis.

ApoE Expressed by Eukaryotic Cell Lines. Human apoE3 or apoE4 wereexpressed individually in RAW 264 mouse macrophage cells (A.T.C.C.TIB71) stably transfected with genomic DNA encoding the human apoEisoforms, and in human embryonic kidney (HEK) 293 cells (A.T.C.C.CRL1573) stably transfected with cDNA encoding the human apoE isoforms,and harvested in serum-free conditioned medium for each cell line asdescribed (LaDu et al., J Biol Chem 1994; 269:23403-6; LaDu et al., JBiol Chem 1995; 270:9039-42; Smith et al., J Biol Chem 1988; 263:8300-8;Miyata et al., Nat Genet 1996; 14:55-61). Concentrations of secretedapoE were determined by capture-ELISA (ApoTek ApoE; PerImmune,Rockville. MD, U.S.A.) after incubating the harvested conditioned mediawith 0.05% β-octyl glucopyranoside for 1 h. Aliquots of conditionedmedia containing apoE3 or apoE4 were stored at 4° C. and used within 2weeks of harvesting.

Purification of apoE from RAW-264-Cell Conditioned Media. PolyclonalAB947 anti-apoE antibody (1 ml. Chemicon, Temecula, Calif., U.S.A.) wascoupled to 2 ml of CNBr-activated Sepharose 4B according to themanufacturer's instructions. Conditioned media containing apoE3 or apoE4was loaded on to the AB947-affinity matrix. Bound apoE was eluted with0.2M acetic acid, pH 2.2, and immediately neutralized. The elutionprofile was monitored at 280 nm and the pertinent fractions were pooledand dialysed against 0.02 M Tris/HCl, pH 8.5, containing 0.1 M NaCl.

Isolation of apoE-Containing Particles from HEK-293-Cell ConditionedMedia. Aliquots of serum-free conditioned media from HEK-293 cellsstably transfected with human apoE3 or apoE4 cDNA, in which theapolipoproteins constituted approximately 50% of the total proteincontent, were concentrated 50-fold with Centricon-10 (Amicon; Millipore,Bedford, Mass., U.S.A.) as described previously. Particles thatcontained apoE3 or apoE4 were isolated from the correspondingconcentrated conditioned media by FPLC using tandem Superose 6 HR 10/30columns (Pharmacia, Piscataway, N.J., U.S.A.) equilibrated in 0.02 Msodium phosphage, pH 7.4, containing 0.05 M NaCl, 0.03% EDTA and 0.02%sodium azide.

Delipidation of apoE Isoforms Purified from Conditioned Media ofEukaryotic Cell Lines and from Baculovirus-Transfected Sf9 Cells. WhenapoE is made recombinantly, it is delipidated. This form of apoE isavailable commercially. Under physiological conditions or uponincorporation into r-HDL particles it becomes lipidated. ApoE in bothforms binds Aβ with high affinity.

ApoE isoforms from RAW-264 and HEK-293 cells, purified as describedabove, as well as apoE produced in Sf9 insect cells by the baculovirusexpression system (PanVera), were delipidated in aqueous state usingdiethyl ether and ethanol. Briefly, the lipoprotein-containing sampleswere extracted with an equal volume of a 3:2 (v/v) diethyl ether/ethanolmixture, followed by four subsequent extractions of the aqueous phasewith a 3:1 (v/v) diethyl ether/ethanol solution. After the finalextraction, the remaining solvent was evaporated under a N2 stream andthe apoE concentration determined as described above.

Incorporation of apoE Into Reconstituted High-Density Lipoprotein (rHDL)Particles. Total lipids were extracted from the human HDL fraction usingthe following method. Human HDL fractions were isolated by preparativegradient ultracentrifugation of plasma obtained from normal healthysubjects, ages 25-40, after a 10-12 h. fast. The HDL fractions weredialysed extensively at 4° C. against PBS containing 1 mM EDTA, and thetotal lipid fractions (HDL-lipid) were extracted with a mixture ofchloroform and methanol (1:2, v/v) and centrifuged at 1700 g for 5 min.The bottom layer that contained the extracted lipids was collected,dried tinder a N2 atmosphere, dissolved in chloroform and stored at −70°C. until use. The amount of total cholesterol, total triacylglycerolsand phospholipids in the HDL and HDL-lipid fractions were determinedenzymically with Sigma (St. Louis, Mo., U.S.A.) diagnostic kits.

Reconstituted apoE-containing HDL particles were prepared as follows.rHDL particles containing apoE were prepared as described usingrecombinant apoE expressed in baculovirus-infected SP) cells and theHDL-lipids extracted from human plasma HDL lipoparticles. In a typicalexperiment, the HDL-lipids (500 μg) were placed in a glass tube, driedunder N2 atmosphere, resuspended in 0.01 M Tris/HCl buffer, pH 8,containing 0.15 M NaCl (TBS) and 0.001 M EDTA. After the addition of 280μg of sodium cholate, the suspension was incubated at 4° C. for 12 h.Subsequently, 500 μg of either St) apoE3 or apoE4 was added to thereaction, incubated at 4° C. for another 12 h, and dialysed extensivelyat 4° C. against PBS containing 0.01% EDTA. The fractions containingapoE incorporated into lipoparticles (apoE-rHDL) were separated fromlipid-free apoE by gel-filtration chromatography using a SUPEROSE 12®column (Pharmacia) equilibrated in 0.02 M phosphate buffer, pH 7.4,containing 0.05M NaCl, 0.03% EDTA and 0.02% sodium azide, at a now rateof 0.8 ml/min. Collected fractions were analyzed by native PAGE using4-20% Tris/glycine gels and Western-blot analysis employing a monoclonalanti-apoE antibody (3D12; BioDesign, Kennebunk, Me., U.S.A.). Thefractions containing apoE-rHDL were pooled for solid-phase bindingstudies and the concentration of apoE in the lipoparticles wasdetermined using the ApoTek ApoE system as described above.

rHDL-particles were chemically crosslinked as follows. apoE moleculesreconstituted into HDL particles were cross-linked usingbis(sulphosuccinimidyl) suberate (BS3). Briefly, BS3 was added to theapoE-rHDL fraction at a concentration of 0.002 M in PBS, incubated atroom temperature for 4 h., and the reaction stopped by the addition of0.03 M Tris/HCl buffer, Ph 7.4. After desalting with Microcon 10(Amicon, Millipore) and lyophilization, the cross-linked samples wereseparated by Tris/Tricine PAGE (10% polyacrylamide), transferred on toan Immobilon-P membrane (Millipore) and reacted with monoclonal 3D12anti-apoE antibody followed by horseradish-peroxidase-conjugatedanti-mouse IgG. The Western blot was developed by chemiluminescenceusing the Super-Signal kit (Pierce, Rockford, Ill., U.S.A.).

Solid-Phase Binding Assays. The binding of apoE to Aβ species wasstudied by ELISA using immobilized freshly prepared (non-aggregated) or72-h-aggregated Aβ40 and Aβ42 and apoE3 or apoE4 isoforms with differentdegrees of lipidation. Polystyrene microtitre plates (Immulon2; DynexTechnology, Chantilly, Va., U.S.A.) were coated for 2 h. at 37° C. witheither fresh or aggregated Aβ40 and Aβ42 (400 ng in 100 μl of 0.1 MNaHCO₃, pH 9.6, per well). Under these conditions, 10 ng of fresh Aβ40,9.6 ng of fresh Aβ42, 10.2 ng of aggregated Aβ40 and 10.9 ng ofaggregated Aβ42 (representing 2.5, 2.4, 2.6 and 2.7% of the peptideoffered, respectively) were coated to the microtitre wells, asdetermined by a modification of the Quantigold assay (DiversifiedBiotech, Boston, Mass., U.S.A.) for protein quantification. Afterblocking with Superblock (Pierce), increasing concentrations of apoE(0-150 nM in TBS; 100 μl per well) were added to the Aβ-coated wells andincubated for 3 h. at 37° C. Bound apoE was detected with monoclonalanti-apoE antibody (3D12, 1:1000) followed byalkaline-phosphatase-conjugated goat F(ab′)2 anti-mouse IgG (1:3000;BioSource International, Camarillo, Calif., U.S.A.). The reaction wasdeveloped for 30 min. with p-nitrophenyl phosphate in diethanolaminebuffer (Bio-Rad, Hercules, Calif., U.S.A.), and quantified at 405 nm ona Microplate Reader (Cambridge Technology, Watertown, Mass., U.S.A.).For Scatchard analysis, bound apoE values were expressed in fmol withthe aid of a calibration curve in which known concentrations of apoEcoated to microtitre wells (as determined by Quantigold assay) werereacted with 3D12, followed by alkaline-phosphatase-conjugated F(ab′)2anti-mouse IgG under conditions identical with those described above.Under the experimental conditions employed, an excess of apoE wasreacted with solid-phase Aβ; therefore, only a small fraction of addedligand bound to the immobilized peptide and the concentration of freeligand was considered equivalent to the total apoE added.

Results

Increasing concentrations (0-150 nM) of various apoE3 preparations werereacted with microliter ELISA wells coated with non-aggregated Aβ40 orAβ42 for 3 hours. Bound apoE was detected in all cases, with monoclonal3D12 anti-apoE antibody followed by alkaline phosphatase-labeledanti-mouse IgG. The results are shown in FIG. 1.

In FIG. 1, (A, D) Binding to Aβ40 and Aβ42, respectively, of Sf9-derivedapoE3 and apoE4, both delipidated and upon incorporation into r-HDLs.(B, E) Binding to Aβ40 and Aβ42, respectively, of HEK-derived apoE3 andapoE4 both, in their native HDL particles and following delipidation.(C, F) Binding to Aβ40 and Aβ42, respectively, of RAW-derived apoE3 andapoE4, in the native HDL-particles and following delipidation. Eachpoint represents the mean (±standard deviation) of triplicates.

These results show the high affinity binding of apoE to Aβ. Therefore,apoE, and the other compounds disclosed herein, may be used as Aβbinding compounds for the therapeutic purposes disclosed herein.

Example 2 Elicitation of Anti-Aβ-Antibodies Removing Aβ from Blood

This Example describes immunization of animals with syntheticAβ-derivatives, with subsequent analysis of Aβ content in brain andblood after in vivo formation of anti-Aβ-antibodies. According to theinvention, similar results could be obtained by administering externallyproduced antibodies against Aβ.

Briefly, mice received their first immunization withK₆Aβ1-30-NH₂(E₁₈E₁₉) at 10.5-13 months of age (peptide: n=23, vehicle:n=24). The animals were bled prior to vaccination, 3 months followingthe first injection and at the time of sacrifice at 18 to 21 months. Themice were tested in the radial arm maze, subsequently perfused and theirbrains processed as described (Sigurdsson et al., Am. J. Pathol. 2001;159:439-4471) (peptide: n=18; vehicle: n=18).

Materials and Methods

Peptide. K₆Aβ1-30-NH₂(E₁₈E₁₉) was synthesized at the Keck Foundation(Yale University, New Haven, Conn.). It consists of the first 30 aminoacids of amyloid-β with glutamate substituted for valine andphenylalanine in positions 18 and 19. These substitutions result in aT-cell independent immune response as seen by a high IgM response andlow IgG response.

Animals. The vaccination was performed in the Tg2576 APP mouse modeldeveloped by Karen Hsiao and colleagues (Hsiao et al., Science 1996;274:99-1022). These mice develop Aβ plaques as early as at 11 to 13months of age. The animals were maintained on a 12-hour light-darkcycle, and had access to food and water ad libitum. The animal care wasin accordance with institutional guidelines.

Vaccine Administration. K₆Aβ1-30-NH₂(E₁₈E₁₉) was supplied astrifluoroacetic acid salt. The immunization procedure was performed aspreviously described by us (Sigurdsson et al., Am. J. Pathol. 2001;159:439-4471) which is the same protocol as described by Schenk andcolleagues (Schenk et al., Nature 1999; 400:173-1773) except that thepeptide was not incubated overnight at 37° C. before injection. Briefly,the peptide was dissolved in phosphate-buffered saline (PBS) at aconcentration of 2 mg/ml and then mixed 1:1 (v/v) with the adjuvant orPBS. Complete Freund's adjuvant was used for the first injection,incomplete Freund's adjuvant for the next three injections, and PBS fromthe fifth injection forward. The mice received a subcutaneous injectionof 100 μl of the mixture (ie, 100 μg/100 μl) followed by a secondinjection 2 weeks later, and then monthly thereafter. Vaccination usingthe K6Aβ1-30-NH₂(E₁₈E₁₉) peptide started when the mice were 10.5-13months of age and the mice were sacrificed at 18-21 months of age.

Radial Arm Maze. Animals were kept in test room throughout theexperiment, behind a cover to prevent view of the apparatus and room.Each animal underwent 2 days of adaptation, consisting of 15 minutes ofmaze exploration (2 subjects at a time), with 3 pieces of fruit loops ineach arm. Subjects were exposed to arm doors only on day 2. Animals werefood deprived 1 day before the first adaptation session and maintainedat approximately ten percent body weight loss. Fruit loops were added tonormal diet 5 days before deprivation schedule started. Animals enteredand exited the apparatus through the center of the maze. Testingincluded recording correct and incorrect arms entered. Each trial wasinitiated by placing the mouse in the center of the maze and all doorsinto the arms were subsequently opened. After entry into an arm, theanimal had to find and eat the reinforcer before the door was reopenedto allow the animal to re-enter the center of the maze. Testing endedwhen all eight arms had been entered and reinforcers eaten. Re-entryinto an arm constituted an error. Total number of errors and time toenter all eight arms were recorded. The animals were allowed access tofood for up to 3-4 hours daily, depending on their body weight loss. Thecorners and holes in the maze were cleaned with 95% ethanol after eachanimal and the arms

Antibody Levels. Antibody levels were determined by 1:500 dilutions ofplasma using an enzyme-linked immunosorbent assay (ELISA) as describedpreviously (Jimenez-Huete et al., Alzheimers Reports 1998; 1:4147) inwhich Aβ or its derivative is coated onto microliter wells. Theantibodies were detected by a goat anti-mouse IgG linked to ahorseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, N.J.) ora goat anti-mouse IgM peroxidase conjugate (Sigma, A8786), andtetramethyl benzidine (Pierce, Rockford, Ill.) was the substrate.

Histology. Mice were anesthetized with sodium pentobarbital (150 mg/kg,intraperitoneally), perfused transaortically with phosphate buffer andthe brains processed as previously described (Sigurdsson et al.,Neurobiol. Aging 1996; 17:893-901) The right hemisphere was immersionfixed in periodate-lysine-paraformaldehyde (PLP), whereas the lefthemisphere was snap-frozen for measurements of Aβ levels usingestablished ELISA methods (Mehta et al., Arch. Neurol. 2000;57:100-105). Serial coronal sections (40 μm) were cut and every fifthsection was stained with 6E10 which recognizes Aβ and stains bothpre-amyloid and Aβ plaques (Kim et al., Neurosci Res Comm 1990;7:113-122). After sectioning, the series were placed in ethylene glycolcryoprotectant and stored at −20° C. until used. Staining was performedas previously described (Sigurdsson et al., Neurobiol. Aging 1996;17:893-901; Soto et al., Nat Med 1998; 4:822-826). Briefly, sectionswere incubated in 6E10 (kindly provided by Richard Kascsak, Institutefor Basic Research) primary antibody that selectively binds to human Aβat a 1:1000 dilution. A mouse-on-mouse immunodetection kit (VectorLaboratories, Burlingame, Calif.) was used in which the anti-mouse IgGsecondary antibody was used at a 1:2000 dilution. The sections werereacted in 3,3-diaminobenzidine tetrahydrochloride (DAB) with nickelammonium sulfate (Ni; Mallinckrodt, Paris, Ky.) intensification.

Image Analysis. Immunohistochemistry of tissue sections was quantifiedwith a Bioquant image analysis system, and unbiased sampling was used(West et al., Trends Neurosci. 1999; 22:51-61). All procedures wereperformed by an individual blind to the experimental condition of thestudy. Cortical area analyzed was dorsomedially from the cingulatecortex and extended ventrolaterally to the rhinal fissure within theright hemisphere. The area of the grid was 800×800 μm² and amyloid loadwas measured in 20 frames per mouse (each: 640×480 μm²), chosenrandomly. The Aβ burden is defined as the percentage of area in themeasurement field occupied by reaction product. The number of plaqueswere also counted and the plaques were divided into three groups basedon their size (small: 0.01-50 μm²; medium: 50.01-1000 μm²; large: >1000μm²).

Data Analysis. The data for the amyloid burden within the brain wereanalyzed by a Student's i-test, one-tailed (GraphPad Prism). The radialarm maze data was analyzed by two-way ANOVA repeated measures.Bonferroni post hoc test was used to determine if the mice were learningto run the maze. Correlation was determined by calculating the Pearson rcorrelation coefficient.

Results

FIG. 2 shows that K₆Aβ1-30(E₁₈E₁₉)-treated transgenic mice hadsignificantly fewer errors in the radial arm maze compared to theirvehicle-treated controls (two-way ANOVA, repeated measures, p<0.05). Themice that were immunized with the peptide showed improvements on days 3,5, and 7-9 (p<0.01-0.05) compared to their performance on day 1. Thecontrol mice did not improve significantly over time.

FIG. 3 shows that (A) K₆Aβ1-30(E₁₈E₁₉) induced a substantially morepronounced IgM response compared to IgG response as detected in plasmaat 1:500 dilution; and (B) The IgM antibodies generated followingK₆Aβ1-30(E₁₈E₁₉) immunization cross-reacted with Aβ1-40.

FIGS. 4A-4F show that (A, B, C) Immunization with K₆Aβ1-30(E₁₈E₁₉)preferentially reduced small (34% reduction, p=0.02) and medium sizedplaques (29% reduction, p=0.04). Large plaques were not significantly(N.S.) affected. In addition, (D, E, F) low amyloid plaque burdencorrelated with high IgM levels against the immunogen (p<0.05) andAβ1-42 (p=0.05). A trend for correlation was seen for IgM recognizingAβ1-40.

Example 3 Apolipoprotein E3 (ApoE3) Treatment of Patients withAlzheimer's Disease

The present example evaluates the pharmacokinetics and treatment effectsof administered ApoE3 versus placebo adjunctive treatment in patientswith Alzheimer's disease. Either free, recombinantly produced apoEprotein, or apoE protein incorporated into HDL particles can be used inthis method. In some cases, apoE incorporated into HDL particles may bepreferable, as this is the form apoE is generally found in vivo.

Materials and Methods

The study is a randomized, double blind, placebo controlled parallelgroup study, and includes patients meeting the following inclusioncriteria. Participants must either have genetic mutations that willresult in Alzheimer's disease or be diagnosed with the disease, and maybe male or female. Parents or guardians will give informed consent forthose under the age of 18 years. Exclusion criteria include females whoare pregnant or nursing; life-threatening infections; and any conditionmaking participation against the patient's interest.

Patients receive recombinant apoE3 or lipidated recombinant apoE3 as a1-10 mg/ml solution administered intravenously as a single injection orcontinuous drip over several minutes daily or weekly. The dose rangesbetween 0.1 mg and about 10 mg per kg body weight, and the amountadministered ranges between about one-tenth and up to about two-fold ofmean plasma levels of total apoE. The amount administered is preferablynot greater than needed to bind all free Aβ in plasma. Prior totreatment, free plasma Aβ is measured in each patient and theadministered dose calculated based on that data and the patients weight,taking into consideration the known pharmacokinetic properties of apoE3.

Briefly, apoE3 is prepared as described in Example 1. Pharmacokineticsampling is performed with the administration set as time=0. Briefly,blood samples (5 ml) are collected at the following limes: baseline, andat 1, 2, 4, 8, 12, and 24 hours and weekly thereafter. Blood samples arecollected via catheterization of the antecubical or other readilyaccessible vein, or by direct venipuncture. Each tube is mixed andimmediately iced. Plasma is separated within 2 hours by centrifugationat 1500×g for 15 minutes in a refrigerated (4° C.) centrifuge. All tubesare stored at <−20° C. pending analysis. The samples are analyzed forfree and total Aβ levels as well as levels of apoE3. Subsequent dosingwill be based on this information with the aim of maintaining very lowlevels of free Aβ in plasma. Cerebrospinal fluid will be obtained bylumbar puncture at the beginning and end of the study for measurementsof total and free Aβ.

The patients are evaluated prior to treatment and every 6 weeks forsymptoms of Alzheimer's disease using standard cognitive tests asdescribed by, e.g., Rogers et al., Neurology 1998; 50:136-145. Primarymeasures are the Alzheimer's Disease Assessment Scale (ADAS-cog) andClinician's Interview Based Assessment of Change-Plus (CIBIC plus), withMini-Mental State Examination (MMSE), the Clinical Dementia ratingScale-Sum of the Boxes (SDR-SB) and patient rated Quality of Life (QoL)used as secondary measures. Any available imaging method allowingimaging of amyloid burden in live subjects is used concurrently.

The same method can be used to evaluate other Aβ-binding agents setforth in Table 1, with modifications that are within the level of skillin the art, e.g., to modify the amount of active compound to be presentin the plasma so as to compensate for differences in molecular weightbetween apoE and the agent, and for difference in binding affinitytowards Aβ and the agent.

Example 4 Apolipoprotein E3 (ApoE3) Hemodialysis Treatment in Patientswith Alzheimer's Disease

This Example evaluates the treatment effects in patients withAlzheimer's disease of hemodialysis of Aβ from blood by dialyzing theblood through a membrane with apoE3 added to the dialysate bath versusno treatment or versus hemodialysis without apoE3. Either free,recombinantly produced apoE protein, or apoE protein incorporated intoHDL particles can be used in this method, although free apoE protein ispreferred due to the more simple preparation procedure.

Materials and Methods

The study is a randomized, double blind, placebo controlled parallelgroup study, and includes patients meeting the following inclusioncriteria. Participants must either have genetic mutations that willresult in Alzheimer's disease or have been diagnosed with the disease,and may be male or female. Parents or guardians will give informedconsent for those under the age of 18 years. Exclusion criteria includefemales who are pregnant or nursing; life-threatening infections; andany condition making participation against the patient's interest.

Recombinant apoE3 or lipidated recombinant apoE3 is added to thedialysis bath. The concentration does not necessarily have to be greateror different than that needed to bind all free Aβ that diffuses from theblood compartment of the dialysis unit. Prior to treatment, free plasmaAβ is measured in each patient and the dose added to the dialysate bathcalculated based on that data taking into consideration the knownaffinity of apoE3 for Aβ. ApoE3 is prepared as described in Example 1.The semipermeable membrane has a molecular weight cutoff of 10,000Daltons. Soluble free Aβ monomers, dimers and oligomers in the blooddiffuse into the dialysis bath and bind to apoE3. Thereafter, Aβ doesnot diffuse back into the blood.

This procedure is performed monthly with blood samples collected attime=00, immediately following dialysis and at 12, 24 hours and weeklythereafter. Blood samples are collected via catheterization of theantecubical or other readily accessible vein, or by direct venipuncture.Each tube is mixed and immediately iced. Plasma is separated within 2hours by centrifugation at 1500×g for 15 minutes in a refrigerated (4°C.) centrifuge. All tubes are stored at <−20° C. pending analysis. Thesamples are analyzed for free and total Aβ levels. Subsequent dosing isbased on this information with the aim of maintaining very low levels offree Aβ in plasma. Cerebrospinal fluid will be obtained by lumbarpuncture at the beginning and end of the study for measurements of totaland free Aβ.

The patients are evaluated prior to treatment and every 6 weeks forsymptoms of Alzheimer's disease using standard cognitive tests asdescribed by, e.g., Rogers et al. Neurology 1998; 50:136-145. Primarymeasures are the Alzheimer's Disease Assessment Scale (ADAS-cog) andClinician's Interview Based Assessment of Change-Plus (CIBIC plus), withMini-Mental State Examination (MMSE), the Clinical Dementia ratingScale-Sum of the Boxes (SDR-SB) and patient rated Quality of Life (QoL)used as secondary measures. Any available imaging method allowingimaging of amyloid burden in live subjects is used concurrently.

The same method can be used to evaluate other Aβ-binding agents setforth in Table 1, with modifications that are within the level of skillin the art, e.g., to modify the amount of active compound to be presentin the dialysis compartment so as to compensate for differences inmolecular weight between apoE and the agent.

Example 5 Convective Dialysis Treatment in Patients with Alzheimer'sDisease

This Example evaluates the treatment effects in patients withAlzheimer's disease by studying hemofiltration of Aβ from blood bydialyzing the blood through a unidirectional membrane as compared to notreatment.

Materials and Methods

The study is a randomized, double blind, placebo controlled parallelgroup study, and includes patients meeting the following inclusioncriteria. Participants must either have genetic mutations that willresult in Alzheimer's disease or have been diagnosed with the disease,and may be male or female. Parents or guardians will give informedconsent for those under the age of 18 years. Exclusion criteria includefemales who are pregnant or nursing; life-threatening infections; andany condition making participation against the patient's interest.

In this method, there is no need for Aβ binding compounds, and themembranes allow the passage of molecules less than 20,000 Daltons.Filtration across the membrane is unidirectional. Therefore, filtered Aβcannot flow back.

Prior to treatment, free and total plasma Aβ is measured in eachpatient. Soluble free Aβ monomers, dimers and oligomers in the blooddiffuse across the dialysis membrane in response to a transmembranepressure gradient. Thereafter, Aβ will not diffuse back into the blood.

The procedure is performed monthly with blood samples collected attime=0, immediately following dialysis and at 12, 24 hours and weeklythereafter. Blood samples are collected via catheterization of theantecubical or other readily accessible vein, or by direct venipuncture.Each tube is mixed and immediately iced. Plasma is separated within 2hours by centrifugation at 1500×g for 15 minutes in a refrigerated (4°C.) centrifuge. All tubes are stored at <−20° C. pending analysis. Thesamples are analyzed for free and total Aβ levels. The interval betweendialysis treatment is based oil this information with the aim ofmaintaining very low levels of free Aβ in plasma. Cerebrospinal fluidwill be obtained by lumbar puncture at the beginning and end of thestudy for measurements of total and free Aβ.

The patients are evaluated prior to treatment and every 6 weeks forsymptoms of Alzheimer's disease using standard cognitive tests asdescribed by, e.g., Rogers et al., Neurology 1998; 50:136-145. Primarymeasures are the Alzheimer's Disease Assessment Scale (ADAS-cog) andClinician's Interview Based Assessment of Change-Plus (CIBIC plus), withMini-Mental State Examination (MMSE), the Clinical Dementia ratingScale-Sum of the Boxes (SDR-SB) and patient rated Quality of Life (QoL)used as secondary measures. Any available imaging method allowingimaging of amyloid burden in live subjects is used concurrently.

Example 6 Plasma Exchange Dialysis Treatment in Patients withAlzheimer's Disease

This example evaluates the treatment effects in patients withAlzheimer's disease by removal of Aβ from blood by plasma exchangedialysis versus no treatment.

Materials and Methods

The study is a randomized, double blind, placebo controlled parallelgroup study, and includes patients meeting the following inclusioncriteria. Participants must either have genetic mutations that willresult in Alzheimer's disease or have been diagnosed with the disease,and may be male or female. Parents or guardians will give informedconsent for those under the age of 18 years. Exclusion criteria includefemales who are pregnant or nursing; life-threatening infections; andany condition making participation against the patient's interest.

In this method, there is no need for Aβ binding compounds. The plasma isseparated and removed from the rest of the blood to remove Aβcirculating in blood. The red and white blood cells and platelets arereturned to the patient, along with replacement fluid. Using thismethod, all Aβ is removed from the blood. Prior to treatment, free andtotal plasma Aβ would be measured in each patient.

The procedure is performed monthly with blood samples collected attime=0, immediately following dialysis and at 12, 24 hours and weeklythereafter. Blood samples are collected via catheterization of theantecubical or other readily accessible vein, or by direct venipuncture.Each tube is mixed and immediately iced. Plasma is separated within 2hours by centrifugation at 1500×g for 15 minutes in a refrigerated (4°C.) centrifuge. All tubes are stored at <−20° C. pending analysis. Thesamples are analyzed for free and total Aβ levels. The interval betweendialysis treatment is based on this information with the aim ofmaintaining very low levels of free Aβ in plasma. Cerebrospinal fluidwill be obtained by lumbar puncture at the beginning and end of thestudy for measurements of total and free Aβ.

The patients are evaluated prior to treatment and every 6 weeks forsymptoms of Alzheimer's disease using standard cognitive tests asdescribed by, e.g., Rogers et al., Neurology 1998; 50:136-145. Primarymeasures are the Alzheimer's Disease Assessment Scale (ADAS-cog) andClinician's Interview Based Assessment of Change-Plus (CIBIC plus), withMini-Mental State Examination (MMSE), the Clinical Dementia ratingScale-Sum of the Boxes (SDR-SB) and patient rated Quality of Life (QoL)used as secondary measures. Any available imaging method allowingimaging of amyloid burden in live subjects is used concurrently.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying, figures. Such modifications are intended to fall withinthe scope of the appended claims. It is further to be understood thatall values are approximate, and are provided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A method of treating a patient suffering from an amyloid diseasecomprising administering to a patient in need of such treatment atherapeutically effective amount of a compound which binds to freeamyloid-beta in a body fluid of the patient.
 2. The method of claim 1,wherein a binding complex is formed between the compound and Aβ.
 3. Themethod of claim 1, wherein the body fluid is blood.
 4. The method ofclaim 1, wherein the complex is excreted from the patient.
 5. The methodof claim 1, wherein the amyloid disease is Alzheimer's disease.
 6. Themethod of claim 1, wherein the compound is administered systemically. 7.The method of claim 6, wherein between about 1 mg and about 100 mg ofthe compound is administered per kg body weight of the patient and perday.
 8. The method of claim 1, wherein the compound is anamyloid-beta-binding fragment thereof. 9.-12. (canceled)
 13. The methodof claim 1, wherein the compound is an antibody or antibody fragmentwhich binds to amyloid-beta.
 14. The method of claim 1, wherein theblood-brain-barrier is permeabilized prior to administration of thecompound.
 15. The method of claim 14, wherein the blood-brain-barrier ispermeabilized by administering insulin growth factor I (IGF-I). 16.-27.(canceled)